Establishment And Application of An in Vitro Cd8+ T Cell Exhaustion System

Abstract Exhaustion is a dysfunctional state of cytotoxic CD8+ T cells (CTL). In vivo models of CTL exhaustion yield very few exhausted CTL, limiting the analysis can be done on these cells. Furthermore, the milieu in these models can obscure the phenotype of exhausted CTL. An in vitro system could therefore greatly facilitate the study of this cell state. Purified OT-I CTL were cultured and repeatedly stimulated with OVA(257–264) SIINFEKL peptide to induce exhaustion. On day 5, inhibitory receptors (IRs) and cytokine production were assayed. Sorted live CTL were transferred into WSN-OVA influenza infected mice and on day10 post infection the number of the donor CTL were measured. Gene expression changes were determined by RNAseq and DNA methylation sequence. In vitro exhausted CTL exhibited upregulation of multiple IRs as well as impaired polyfunctionality. They expanded less than controls in vivo. In vitro exhausted CTL exhibited all the transcriptomic characteristics of in vivo exhausted CTL. Repeated stimulation of CTL with their specific peptide, induced all the functional, phenotypic and transcriptomic characteristics of exhausted CTL. This in vitro system can be used to identify genes and signalling pathways involved in exhaustion and facilitate the screening of reagents that prevent/reverse CTL exhaustion.